Characterization and Properties of G4X Mutants of Ralstonia eutropha PHA Synthase for Poly(3-hydroxybutyrate) Biosynthesis in Escherichia coli

✍ Scribed by Yahaya M. Normi; Tomohiro Hiraishi; Seiichi Taguchi; Hideki Abe; Kumar Sudesh; Nazalan Najimudin; Yoshiharu Doi

• Book ID: 102468858
• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 263 KB
• Volume: 5
• Category: Article
• ISSN: 1616-5187
• DOI: 10.1002/mabi.200400181

✦ Synopsis

Abstract

Summary: Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC~Re~) was performed through systematic in vitro evolution in order to obtain improved PhaC~Re~ having an enhanced activity of poly(3‐hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N‐terminal mutation important for the enhancement of both PHB content in dry cells and PhaC~Re~ level in vivo was identified. Site‐directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild‐type. In vitro enzymatic assays of purified G4D mutant PhaC~Re~ revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild‐type enzyme.

(A) PHB accumulation and (B) level of PhaC~Re~ in vivo for wild‐type and in particular, the G4D mutant generated in this study.

magnified image(A) PHB accumulation and (B) level of PhaC~Re~ in vivo for wild‐type and in particular, the G4D mutant generated in this study.